보고서 정보
주관연구기관 |
한국기초과학지원연구원 Korea Basic Science Institute |
연구책임자 |
박영목
|
참여연구자 |
유종신
,
김승일
,
남명희
,
김수현
,
권경훈
,
최종순
,
권오옥
,
김영환
,
서종복
,
김진영
,
정영호
,
조건
,
김경욱
,
김은아
,
이정화
,
안영희
,
박은정
,
황수경
,
김수정
,
허은주
,
정미영
|
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2003-08 |
주관부처 |
과학기술부 |
사업 관리 기관 |
한국기초과학지원연구원 Korea Basic Science Institute |
등록번호 |
TRKO200300003812 |
DB 구축일자 |
2013-04-18
|
키워드 |
인삼.모상근.프로테옴.이차원전기영동.MALDI-/ ESI Q-TOF 질량분석기.Ginseng.Hairy root.Proteome.2D gel electrophoresis.MALDI-/ ESI Q-TOE Mass spectrometer.
|
초록
▼
가. 인삼의 프로테옴 추출 방법 및 프로테오믹스 Bioinformatics 체제확립
인삼의 뿌리와 모상근에서 액체질소와 TCA 방법을 이용하여 단백질을 추출하여 한 gel 당 660개 이상의 단백질 spot을 확인할 수 있는 해상도가 매우 높은 gel 이미지를 얻을 수 있었다. (한국조직배양학회지 2001. 11 발표). Mass spec에서 얻어진 data를 이용해 손쉽게 단백질을 동정할 수 있도록 Bioinformatics 방법을 개발하였다. (식물생명공학회지 2002. 9월 논문발표 및 Proteomics 2003. 11
가. 인삼의 프로테옴 추출 방법 및 프로테오믹스 Bioinformatics 체제확립
인삼의 뿌리와 모상근에서 액체질소와 TCA 방법을 이용하여 단백질을 추출하여 한 gel 당 660개 이상의 단백질 spot을 확인할 수 있는 해상도가 매우 높은 gel 이미지를 얻을 수 있었다. (한국조직배양학회지 2001. 11 발표). Mass spec에서 얻어진 data를 이용해 손쉽게 단백질을 동정할 수 있도록 Bioinformatics 방법을 개발하였다. (식물생명공학회지 2002. 9월 논문발표 및 Proteomics 2003. 11)
나. 인삼의 뿌리, 모상근 및 잎의 프로테옴 2D DB 구축
인삼의 뿌리, 모삼근 및 잎에서 프로테옴을 효과적으로 추출하여 재현성 있는 2D를 수행할 수 있는 방법을 확립하였다. 뿌리와 모상근 및 잎에서 프로테옴 연구를 수행하여 PH 3-10, 4-7, 6-11으로 인삼 프로테옴을 전개하여 현재 140여 개의 2D를 구축하였으며 흠페이지(http://polar.kbsi.re.kr/bio/)를 구축하여 사업단 홈페이지에 접속하여 공개하고 있다
다. 인삼의 잎과 모상근에서 발현단백질 분석 및 동정
잎에서 약 230개 단백질 spot을 분석하여 147개의 단백질을 동정하였고, 높은 광량에 의해 조절되는 단백질 (peroxiredoxin과 cytosolic small heat shock protein 둥)을 8개 확인하였다 (Proteomics 2003. 11). 또한 모상근에서 159개의 단백질을 분석하여 96개의 인삼 발현 단백질을 확인하였다 (Proteomics 2003. 11). 이 논문은 인삼을 이용한 프로테옴 분석연구로는 세계 최초의 결과이다.
라. Nano-LC와 ESI Q-TOF MS/MS 분석법 개발
Nano-LC와 ESI-MS/MS 분석법을 이용하여 단백질 내 Lysine의 아세틸화를 정확하게 확인할 수 있는 고감도의 새로운 분석방법을 개발하였다. (Anal. Chem. 논문발표, 2002. 11).
바. 프로테옴 연구를 High throughput analysis를 위한 automation system 확립
Abstract
▼
Panax ginseng C.A. Meyer is a well-known Korean traditional medicine. Until now, even though major research of ginseng has been focused on the pharmacological effect, clinical application and chemical analysis of extracted secondary metabolite for several years, the physiology and gene functions of
Panax ginseng C.A. Meyer is a well-known Korean traditional medicine. Until now, even though major research of ginseng has been focused on the pharmacological effect, clinical application and chemical analysis of extracted secondary metabolite for several years, the physiology and gene functions of ginseng were not well known. In this research, we have developed the protein extraction methods of ginseng root and hairy root for proteome analysis in order to elucidate the gene(s) function of ginseng. Using the liquid nitrogen & TCA method as protein extraction method, about 660 protein spots were detected on the 2-DE gel of hairy root. Additionally, comparative analysis result of 2- DEs of ginseng root & hairy root suggested that proteomes of same organism could be changeable according to the culture condition, growth stages and other stimulus
As an initial step to the comprehensive proteomic analysis of Panax ginseng C. A. Meyer, protein mixtures extracted from the cultured hairy root of Panax ginseng were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE). The protein spots were analyzed and identified by peptide finger printing and internal amino acid sequencing by MALDI-TOF mass spectrometry (MALDI-MS)and ESI-Q TOF mass spectrometry (ESI-MS), respectively. More than 300 protein spots were detected on silver stained 2D gels using at pH 3-10, 4-7, and a 4.5-5.5 gradient. 159 major protein spots were analyzed by peptide fingerprinting or de novosequencing and functions of 91 proteins were identified. Protein identification was achieved using the EST database of Panax ginseng and the protein database of plants like Arabidopsis thaliana and Oryza sativa. However, peptide mass fingerprinting by MALDI-MS was insufficient because of a lack of a genome database for Panax ginseng. Only .17 of 159 protein spots were verified by peptide mass fingerprinting using MALDI-MS whereas 87 out of 102 protein spots, which included 13 of 17 proteins identified by MALDI-MS, were identified by internal amino acid sequencing using MS/MS analysis by ESI-MS. When the internal amino acid sequences were used as identification markers, the identification rate exceeded 85.3%, suggesting that the combination of internal sequencing and EST data analysis was the efficient identification method for proteome analysis of plant having incomplete genome data like ginseng. 2D patterns of the main root and leaves of Panax ginseng differed from that of cultured hairy root, suggesting that proteins are exclusively expressed by different tissues for specific cellular functions. Proteome analysis will undoubtedly be helpful for understanding the physiology of Panax Ginseng.
The most abundant root proteins of Panax ginseng C.A. Meyerhave been detected and identified from comparative proteome analysis with cultured hairy root of Panax ginseng. Four abundant proteins (28, 26, 21 and 20kDa) of Panax ginseng have isoforms with different pI values ontwo-dimensional gel electrophoresis (2-DE). But the results of N-terminal and internal amino acid sequencing showed that all of them originate from a 28 kDa protein, known as ginseng major protein (GMP). The GMP gene was searched for in the EST database of Panax ginsengand found to encode a 27.3 kDa protein having 238 amino acid residues. Analysis of the amino acid sequences indicated that GMP exhibits high sequence homology with plant RNases and RNase-like proteins. However, purified GMP had not RNase activity even though it has conserved amino acid residues known to be essential for active sites of RNase. The GMPs present in Panax ginseng main root was not expressed in cultured hairy roots of Panax ginseng. 2- DE analysis showed that the amounts of GMPs in main roots change according to seasonal fluctuation. These results suggest that the GMPs are root specific RNase-like proteins, which function as vegetative storage proteins of Panax ginseng for survival in the natural environment.
Light intensity is very important factor for the growth of ginseng. To understand the physiological response of ginseng leaves to HL at protein level, we analyzed the proteome of ginseng (Panax ginseng C.A. Meyer) leaves. As a first step, we analyzed proteins spots to make a physiological marker. Protein mixtures were extracted from leaves of ginseng and separated by two-dimensional gel electrophoresis. Identification of protein spots was done by peptide mass tagging analysis using ESI -Q TOF mass spectrometry in conjunction with ginseng EST data and other plant protein databases. Of the 144 protein spots identified, 78spots were identified using protein databases. Of the 44 spots were also identified using ginseng EST data. Additional 66 protein spots were identified using ginseng EST data. Including the proteins identified using protein database, total 66 % of proteins were identified using EST data, and this results show that using ginseng EST database in conjunction with peptide mass tagging can be a useful method for identifying ginseng proteins. Large proportions of identified proteins were chloroplast proteins. For analysis of HL responsive proteins, ginseng leaf proteins were prepared from leaves treated with HL for 0 to 4 hr and visualizedon 2D gels. Six HL responsive proteins were revealed by this work up-regulation of small heat shock protein and cytosolic ascorbate peroxidase and major latex like proteins, and down regulation of putative 3-beta hydroxysteroid dehydrogenase/isomerase like protein, and OEC 23 like protein. Concerning the change of these proteins, protective mechanism of ginseng will be discussed.
Post-translational acetylation of proteins regulates many diverse functions, including DNA recognition, protein-protein interaction and protein stability. The identification of enzymes that regulate protein acetylation has revealed broader use of this modification than was previously suspected. In this study, we describe a method for identifying protein acetylation at lysine residues by analysis of digested protein using HPLC/ESI/MS with a new modification-specific marker ion. Collision induced dissociation with capillary or nano-LC/ESI/TOF-MS was used to obtain a fragment ion useful as a marker for acetylated lysine. Although the acetylated lysine immonium ion at m/z 143.1 has been used as a marker ion for detecting acetylated lysine, it can be confused with internal fragment ion in some peptides, producing false positive results. We have found a novel marker ion at m/z 126.1, which is a further fragment ion induced by the loss of NH3 from the acetylated lysine immonium ions at m/z 143.1. This novel marker ion was found to be more specific and approximately nine times more sensitive than the immonium ion at m/z 143.1. In addition, no interfering ions for acetylated peptides were found in the extracted ion chromatogram at m/z 126.1. The utility of this method was demonstrated with acetylated cytochrome C as a model compound. After the modification was probed by the new marker ion, the acetylated lysine site was determined by the CID-MS spectrum. This method was applied to identify histone H4 acetylation in HeLa cells treated with trichostatin A. Three protein bands separated by acid-urea-triton gel electrophoresis were confirmed as tetra, tri and di-acetylated histone H4 at lysines 5, 8, 12 and 16. This method may be useful for assaying for lysine acetylation, which is an important regulatory process for a range of biological functions.
목차 Contents
- Part 1. 인삼 모상근의 인삼 표준 Map 작성 및 발현단백질의 기능분석...20
- Part 2. 프로테옴을 이용한 인삼 잎의 엽소병 관련 단백질 규명...66
- Part 3. Proteome informatics를 이용한 인삼프로테옴분석 - EST data와 질량분석 data 중심으로 -...110
- Part 4. Capillary와 Nano-LC/ESI-MS/MS)를 이용한 수식화된 단백질 중 아세틸 lysine의 확인을 위한 분석방법 및 H4 단백질에서 아세 틸화의 확인...162
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